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United States Patent | 5,747,287 |
Blake ,   et al. | May 5, 1998 |
The present invention relates, in general, to a method for the high level expression of the outer membrane protein meningococcal group B porin proteins and fusion proteins thereof. In particular, the present invention relates to a method of expressing the outer membrane protein meningococcal group B porin proteins in E. coli wherein the meningococcal group B porin proteins and fusion proteins thereof comprise more than 2% of the total protein expressed in E. coli. The invention also relates to a method of purification and refolding of the meningococcal group B porin proteins and fusion proteins thereof and to their use in vaccines.
Inventors: | Blake; Milan S. (New York, NY); Tai; Joseph Y. (Fort Washington, PA); Qi; Huilin L. (New York, NY); Liang; Shu-Mei (Bethesda, MD); Hronowski; Lucjan J. J. (Laurel, MD); Pullen; Jeffrey K. (Columbia, MD) |
Assignee: | North American Vaccine, Inc. (Beltsville, MD); The Rockefeller University (New York, NY) |
Appl. No.: | 877109 |
Filed: | June 17, 1997 |
Current U.S. Class: | 435/69.1; 424/193.1; 424/249.1; 435/69.3; 435/69.7; 435/252.33; 435/320.1; 530/350 |
Intern'l Class: | C07K 001/00; C12P 021/06; C12N 015/00; A61K 039/05 |
Field of Search: | 435/69.1,69.3,69.7,172.3,252.33,320.1 530/350 424/193.1,249.1 |
4134214 | Jan., 1979 | Graham et al. | 34/5. |
4271147 | Jun., 1981 | Helting et al. | 424/92. |
4356170 | Oct., 1982 | Jennings et al. | 424/92. |
4451446 | May., 1984 | Vandevelde et al. | 424/92. |
4727136 | Feb., 1988 | Jennings et al. | 530/395. |
5425946 | Jun., 1995 | Tai et al. | 424/197. |
5576002 | Nov., 1996 | Jennings et al. | 424/197. |
5623057 | Apr., 1997 | Marburg et al. | 530/404. |
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______________________________________ Grow organisms in modified Franz medium 10 to 20 hrs .dwnarw. Heat kill, 55.degree. C., 10 min Remove inactivated cells by centrifugation .dwnarw. Add Cetavlon to 0.1% Precipitate CP from culture broth .dwnarw. Add calcium chloride to 1M Dissolve CP then centrifuge to remove cellular debris .dwnarw. Add ethyl alcohol to 25% Remove precipitated nucleic acids by centrifugation .dwnarw. Add ethyl alcohol to 80% Precipitate crude CP and remove alcohol ______________________________________
TABLE 1 ______________________________________ ELISA and Bactericidal Titers of Group B Meningococcal Conjugate Vaccines (N-Pr GBMP-Protein) Bactericidal Vaccine Adjuvant ELISA Titer Titer ______________________________________ N-Pr GBMP-TT Saline 5,400 0 Al(OH).sub.3 13,000 0 ST.sup.1 17,000 0 CFA.sup.2 40,000 800 N-Pr GBMP-PP Saline 20,000 500 Saline 22,000 150 Saline 39,000 960 Al(OH).sub.3 93,000 200 Al(OH).sub.3 166,000 >3,200 Al(OH).sub.3 130,000 1,200 ST 53,000 1,000 ST 29,000 1,700 ST 72,000 1,500 N-Pr GBMP Saline >100 0 Al(OH).sub.3 >100 0 ST >100 0 PP Saline >100 0 Al(OH).sub.3 >100 0 ST 660 0 ______________________________________ .sup.1 ST = Stearyl tyrosine. .sup.2 CFA = Complete Freund's Adjuvant
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 23 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 930 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..930 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTGTACGGTACAATTAAAGCAGGCGTAGAAACTTCCCGCTCTGTATTT48 LeuTyrGlyThrIleLysAlaGlyValGluThrSerArgSerValPhe 151015 CACCAGAACGGCCAAGTTACTGAAGTTACAACCGCTACCGGCATCGTT96 HisGlnAsnGlyGlnValThrGluValThrThrAlaThrGlyIleVal 202530 GATTTGGGTTCGAAAATCGGCTTCAAAGGCCAAGAAGACCTCGGTAAC144 AspLeuGlySerLysIleGlyPheLysGlyGlnGluAspLeuGlyAsn 354045 GGCCTGAAAGCCATTTGGCAGGTTGAGCAAAAAGCATCTATCGCCGGT192 GlyLeuLysAlaIleTrpGlnValGluGlnLysAlaSerIleAlaGly 505560 ACTGACTCCGGTTGGGGCAACCGCCAATCCTTCATCGGCTTGAAAGGC240 ThrAspSerGlyTrpGlyAsnArgGlnSerPheIleGlyLeuLysGly 65707580 GGCTTCGGTAAATTGCGCGTCGGTCGTTTGAACAGCGTCCTGAAAGAC288 GlyPheGlyLysLeuArgValGlyArgLeuAsnSerValLeuLysAsp 859095 ACCGGCGACATCAATCCTTGGGATAGCAAAAGCGACTATTTGGGTGTA336 ThrGlyAspIleAsnProTrpAspSerLysSerAspTyrLeuGlyVal 100105110 AACAAAATTGCCGAACCCGAGGCACGCCTCATTTCCGTACGCTACGAT384 AsnLysIleAlaGluProGluAlaArgLeuIleSerValArgTyrAsp 115120125 TCTCCCGAATTTGCCGGCCTCAGCGGCAGCGTACAATACGCGCTTAAC432 SerProGluPheAlaGlyLeuSerGlySerValGlnTyrAlaLeuAsn 130135140 GACAATGCAGGCAGACATAACAGCGAATCTTACCACGCCGGCTTCAAC480 AspAsnAlaGlyArgHisAsnSerGluSerTyrHisAlaGlyPheAsn 145150155160 TACAAAAACGGTGGCTTCTTCGTGCAATATGGCGGTGCCTATAAAAGA528 TyrLysAsnGlyGlyPhePheValGlnTyrGlyGlyAlaTyrLysArg 165170175 CATCATCAAGTGCAAGAGGGCTTGAATATTGAGAAATACCAGATTCAC576 HisHisGlnValGlnGluGlyLeuAsnIleGluLysTyrGlnIleHis 180185190 CGTTTGGTCAGCGGTTACGACAATGATGCCCTGTACGCTTCCGTAGCC624 ArgLeuValSerGlyTyrAspAsnAspAlaLeuTyrAlaSerValAla 195200205 GTACAGCAACAAGACGCGAAACTGACTGATGCTTCCAATTCGCACAAC672 ValGlnGlnGlnAspAlaLysLeuThrAspAlaSerAsnSerHisAsn 210215220 TCTCAAACCGAAGTTGCCGCTACCTTGGCATACCGCTTCGGCAACGTA720 SerGlnThrGluValAlaAlaThrLeuAlaTyrArgPheGlyAsnVal 225230235240 ACGCCCCGAGTTTCTTACGCCCACGGCTTCAAAGGTTTGGTTGATGAT768 ThrProArgValSerTyrAlaHisGlyPheLysGlyLeuValAspAsp 245250255 GCAGACATAGGCAACGAATACGACCAAGTGGTTGTCGGTGCGGAATAC816 AlaAspIleGlyAsnGluTyrAspGlnValValValGlyAlaGluTyr 260265270 GACTTCTCCAAACGCACTTCTGCCTTGGTTTCTGCCGGTTGGTTGCAA864 AspPheSerLysArgThrSerAlaLeuValSerAlaGlyTrpLeuGln 275280285 GAAGGCAAAGGCGAAAACAAATTCGTAGCGACTGCCGGCGGTGTTGGT912 GluGlyLysGlyGluAsnLysPheValAlaThrAlaGlyGlyValGly 290295300 CTGCGTCACAAATTCTAA930 LeuArgHisLysPhe 305 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 309 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: LeuTyrGlyThrIleLysAlaGlyValGluThrSerArgSerValPhe 151015 HisGlnAsnGlyGlnValThrGluValThrThrAlaThrGlyIleVal 202530 AspLeuGlySerLysIleGlyPheLysGlyGlnGluAspLeuGlyAsn 354045 GlyLeuLysAlaIleTrpGlnValGluGlnLysAlaSerIleAlaGly 505560 ThrAspSerGlyTrpGlyAsnArgGlnSerPheIleGlyLeuLysGly 65707580 GlyPheGlyLysLeuArgValGlyArgLeuAsnSerValLeuLysAsp 859095 ThrGlyAspIleAsnProTrpAspSerLysSerAspTyrLeuGlyVal 100105110 AsnLysIleAlaGluProGluAlaArgLeuIleSerValArgTyrAsp 115120125 SerProGluPheAlaGlyLeuSerGlySerValGlnTyrAlaLeuAsn 130135140 AspAsnAlaGlyArgHisAsnSerGluSerTyrHisAlaGlyPheAsn 145150155160 TyrLysAsnGlyGlyPhePheValGlnTyrGlyGlyAlaTyrLysArg 165170175 HisHisGlnValGlnGluGlyLeuAsnIleGluLysTyrGlnIleHis 180185190 ArgLeuValSerGlyTyrAspAsnAspAlaLeuTyrAlaSerValAla 195200205 ValGlnGlnGlnAspAlaLysLeuThrAspAlaSerAsnSerHisAsn 210215220 SerGlnThrGluValAlaAlaThrLeuAlaTyrArgPheGlyAsnVal 225230235240 ThrProArgValSerTyrAlaHisGlyPheLysGlyLeuValAspAsp 245250255 AlaAspIleGlyAsnGluTyrAspGlnValValValGlyAlaGluTyr 260265270 AspPheSerLysArgThrSerAlaLeuValSerAlaGlyTrpLeuGln 275280285 GluGlyLysGlyGluAsnLysPheValAlaThrAlaGlyGlyValGly 290295300 LeuArgHisLysPhe 305 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1029 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1029 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGGACGTTACCTTGTACGGTACAATTAAAGCAGGCGTAGAAGTTTCT48 MetAspValThrLeuTyrGlyThrIleLysAlaGlyValGluValSer 151015 CGCGTAAAAGATGCTGGTACATATAAAGCTCAAGGCGGAAAATCTAAA96 ArgValLysAspAlaGlyThrTyrLysAlaGlnGlyGlyLysSerLys 202530 ACTGCAACCCAAATTGCCGACTTCGGTTCTAAAATCGGTTTCAAAGGT144 ThrAlaThrGlnIleAlaAspPheGlySerLysIleGlyPheLysGly 354045 CAAGAAGACCTCGGCAACGGCATGAAAGCCATTTGGCAGTTGGAACAA192 GlnGluAspLeuGlyAsnGlyMetLysAlaIleTrpGlnLeuGluGln 505560 AAAGCCTCCATCGCCGGCACTAACAGCGGCTGGGGTAACCGCCAGTCC240 LysAlaSerIleAlaGlyThrAsnSerGlyTrpGlyAsnArgGlnSer 65707580 TTCATCGGCTTGAAAGGCGGCTTCGGTACCGTCCGCGCCGGTAATCTG288 PheIleGlyLeuLysGlyGlyPheGlyThrValArgAlaGlyAsnLeu 859095 AACACCGTATTGAAAGACAGCGGCGACAACGTCAATGCATGGGAATCT336 AsnThrValLeuLysAspSerGlyAspAsnValAsnAlaTrpGluSer 100105110 GGTTCTAACACCGAAGATGTACTGGGACTGGGTACTATCGGTCGTGTA384 GlySerAsnThrGluAspValLeuGlyLeuGlyThrIleGlyArgVal 115120125 GAAAGCCGTGAAATCTCCGTACGCTACGACTCTCCCGTATTTGCAGGC432 GluSerArgGluIleSerValArgTyrAspSerProValPheAlaGly 130135140 TTCAGCGGCAGCGTACAATACGTTCCGCGCGATAATGCGAATGATGTG480 PheSerGlySerValGlnTyrValProArgAspAsnAlaAsnAspVal 145150155160 GATAAATACAAACATACGAAGTCCAGCCGTGAGTCTTACCACGCCGGT528 AspLysTyrLysHisThrLysSerSerArgGluSerTyrHisAlaGly 165170175 CTGAAATACGAAAATGCCGGTTTCTTCGGTCAATACGCAGGTTCTTTT576 LeuLysTyrGluAsnAlaGlyPhePheGlyGlnTyrAlaGlySerPhe 180185190 GCCAAATATGCTGATTTGAACACTGATGCAGAACGTGTTGCAGTAAAT624 AlaLysTyrAlaAspLeuAsnThrAspAlaGluArgValAlaValAsn 195200205 ACTGCAAATGCCCATCCTGTTAAGGATTACCAAGTACACCGCGTAGTT672 ThrAlaAsnAlaHisProValLysAspTyrGlnValHisArgValVal 210215220 GCCGGTTACGATGCCAATGACCTGTACGTTTCTGTTGCCGGTCAGTAT720 AlaGlyTyrAspAlaAsnAspLeuTyrValSerValAlaGlyGlnTyr 225230235240 GAAGCTGCTAAAAACAACGAGGTTGGTTCTACCAAGGGTAAAAAACAC768 GluAlaAlaLysAsnAsnGluValGlySerThrLysGlyLysLysHis 245250255 GAGCAAACTCAAGTTGCCGCTACTGCCGCTTACCGTTTTGGCAACGTA816 GluGlnThrGlnValAlaAlaThrAlaAlaTyrArgPheGlyAsnVal 260265270 ACGCCTCGCGTTTCTTACGCCCACGGCTTCAAAGCTAAAGTGAATGGC864 ThrProArgValSerTyrAlaHisGlyPheLysAlaLysValAsnGly 275280285 GTGAAAGACGCAAATTACCAATACGACCAAGTTATCGTTGGTGCCGAC912 ValLysAspAlaAsnTyrGlnTyrAspGlnValIleValGlyAlaAsp 290295300 TACGACTTCTCCAAACGCACTTCCGCTCTGGTTTCTGCCGGTTGGTTG960 TyrAspPheSerLysArgThrSerAlaLeuValSerAlaGlyTrpLeu 305310315320 AAACAAGGTAAAGGCGCGGGAAAAGTCGAACAAACTGCCAGCATGGTT1008 LysGlnGlyLysGlyAlaGlyLysValGluGlnThrAlaSerMetVal 325330335 GGTCTGCGTCACAAATTCTAA1029 GlyLeuArgHisLysPhe 340 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 342 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: MetAspValThrLeuTyrGlyThrIleLysAlaGlyValGluValSer 151015 ArgValLysAspAlaGlyThrTyrLysAlaGlnGlyGlyLysSerLys 202530 ThrAlaThrGlnIleAlaAspPheGlySerLysIleGlyPheLysGly 354045 GlnGluAspLeuGlyAsnGlyMetLysAlaIleTrpGlnLeuGluGln 505560 LysAlaSerIleAlaGlyThrAsnSerGlyTrpGlyAsnArgGlnSer 65707580 PheIleGlyLeuLysGlyGlyPheGlyThrValArgAlaGlyAsnLeu 859095 AsnThrValLeuLysAspSerGlyAspAsnValAsnAlaTrpGluSer 100105110 GlySerAsnThrGluAspValLeuGlyLeuGlyThrIleGlyArgVal 115120125 GluSerArgGluIleSerValArgTyrAspSerProValPheAlaGly 130135140 PheSerGlySerValGlnTyrValProArgAspAsnAlaAsnAspVal 145150155160 AspLysTyrLysHisThrLysSerSerArgGluSerTyrHisAlaGly 165170175 LeuLysTyrGluAsnAlaGlyPhePheGlyGlnTyrAlaGlySerPhe 180185190 AlaLysTyrAlaAspLeuAsnThrAspAlaGluArgValAlaValAsn 195200205 ThrAlaAsnAlaHisProValLysAspTyrGlnValHisArgValVal 210215220 AlaGlyTyrAspAlaAsnAspLeuTyrValSerValAlaGlyGlnTyr 225230235240 GluAlaAlaLysAsnAsnGluValGlySerThrLysGlyLysLysHis 245250255 GluGlnThrGlnValAlaAlaThrAlaAlaTyrArgPheGlyAsnVal 260265270 ThrProArgValSerTyrAlaHisGlyPheLysAlaLysValAsnGly 275280285 ValLysAspAlaAsnTyrGlnTyrAspGlnValIleValGlyAlaAsp 290295300 TyrAspPheSerLysArgThrSerAlaLeuValSerAlaGlyTrpLeu 305310315320 LysGlnGlyLysGlyAlaGlyLysValGluGlnThrAlaSerMetVal 325330335 GlyLeuArgHisLysPhe 340 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1092 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant (ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..1092 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATTCAAGCTTG48 MetAlaSerMetThrGlyGlyGlnGlnMetGlyArgAspSerSerLeu 151015 GTACCGAGCTCGGATCCAGACGTTACCTTGTACGGTACAATTAAAGCA96 ValProSerSerAspProAspValThrLeuTyrGlyThrIleLysAla 202530 GGCGTAGAAGTTTCTCGCGTAAAAGATGCTGGTACATATAAAGCTCAA144 GlyValGluValSerArgValLysAspAlaGlyThrTyrLysAlaGln 354045 GGCGGAAAATCTAAAACTGCAACCCAAATTGCCGACTTCGGTTCTAAA192 GlyGlyLysSerLysThrAlaThrGlnIleAlaAspPheGlySerLys 505560 ATCGGTTTCAAAGGTCAAGAAGACCTCGGCAACGGCATGAAAGCCATT240 IleGlyPheLysGlyGlnGluAspLeuGlyAsnGlyMetLysAlaIle 65707580 TGGCAGTTGGAACAAAAAGCCTCCATCGCCGGCACTAACAGCGGCTGG288 TrpGlnLeuGluGlnLysAlaSerIleAlaGlyThrAsnSerGlyTrp 859095 GGTAACCGCCAGTCCTTCATCGGCTTGAAAGGCGGCTTCGGTACCGTC336 GlyAsnArgGlnSerPheIleGlyLeuLysGlyGlyPheGlyThrVal 100105110 CGCGCCGGTAATCTGAACACCGTATTGAAAGACAGCGGCGACAACGTC384 ArgAlaGlyAsnLeuAsnThrValLeuLysAspSerGlyAspAsnVal 115120125 AATGCATGGGAATCTGGTTCTAACACCGAAGATGTACTGGGACTGGGT432 AsnAlaTrpGluSerGlySerAsnThrGluAspValLeuGlyLeuGly 130135140 ACTATCGGTCGTGTAGAAAGCCGTGAAATCTCCGTACGCTACGACTCT480 ThrIleGlyArgValGluSerArgGluIleSerValArgTyrAspSer 145150155160 CCCGTATTTGCAGGCTTCAGCGGCAGCGTACAATACGTTCCGCGCGAT528 ProValPheAlaGlyPheSerGlySerValGlnTyrValProArgAsp 165170175 AATGCGAATGATGTGGATAAATACAAACATACGAAGTCCAGCCGTGAG576 AsnAlaAsnAspValAspLysTyrLysHisThrLysSerSerArgGlu 180185190 TCTTACCACGCCGGTCTGAAATACGAAAATGCCGGTTTCTTCGGTCAA624 SerTyrHisAlaGlyLeuLysTyrGluAsnAlaGlyPhePheGlyGln 195200205 TACGCAGGTTCTTTTGCCAAATATGCTGATTTGAACACTGATGCAGAA672 TyrAlaGlySerPheAlaLysTyrAlaAspLeuAsnThrAspAlaGlu 210215220 CGTGTTGCAGTAAATACTGCAAATGCCCATCCTGTTAAGGATTACCAA720 ArgValAlaValAsnThrAlaAsnAlaHisProValLysAspTyrGln 225230235240 GTACACCGCGTAGTTGCCGGTTACGATGCCAATGACCTGTACGTTTCT768 ValHisArgValValAlaGlyTyrAspAlaAsnAspLeuTyrValSer 245250255 GTTGCCGGTCAGTATGAAGCTGCTAAAAACAACGAGGTTGGTTCTACC816 ValAlaGlyGlnTyrGluAlaAlaLysAsnAsnGluValGlySerThr 260265270 AAGGGTAAAAAACACGAGCAAACTCAAGTTGCCGCTACTGCCGCTTAC864 LysGlyLysLysHisGluGlnThrGlnValAlaAlaThrAlaAlaTyr 275280285 CGTTTTGGCAACGTAACGCCTCGCGTTTCTTACGCCCACGGCTTCAAA912 ArgPheGlyAsnValThrProArgValSerTyrAlaHisGlyPheLys 290295300 GCTAAAGTGAATGGCGTGAAAGACGCAAATTACCAATACGACCAAGTT960 AlaLysValAsnGlyValLysAspAlaAsnTyrGlnTyrAspGlnVal 305310315320 ATCGTTGGTGCCGACTACGACTTCTCCAAACGCACTTCCGCTCTGGTT1008 IleValGlyAlaAspTyrAspPheSerLysArgThrSerAlaLeuVal 325330335 TCTGCCGGTTGGTTGAAACAAGGTAAAGGCGCGGGAAAAGTCGAACAA1056 SerAlaGlyTrpLeuLysGlnGlyLysGlyAlaGlyLysValGluGln 340345350 ACTGCCAGCATGGTTGGTCTGCGTCACAAATTCTAA1092 ThrAlaSerMetValGlyLeuArgHisLysPhe 355360 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 363 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: MetAlaSerMetThrGlyGlyGlnGlnMetGlyArgAspSerSerLeu 151015 ValProSerSerAspProAspValThrLeuTyrGlyThrIleLysAla 202530 GlyValGluValSerArgValLysAspAlaGlyThrTyrLysAlaGln 354045 GlyGlyLysSerLysThrAlaThrGlnIleAlaAspPheGlySerLys 505560 IleGlyPheLysGlyGlnGluAspLeuGlyAsnGlyMetLysAlaIle 65707580 TrpGlnLeuGluGlnLysAlaSerIleAlaGlyThrAsnSerGlyTrp 859095 GlyAsnArgGlnSerPheIleGlyLeuLysGlyGlyPheGlyThrVal 100105110 ArgAlaGlyAsnLeuAsnThrValLeuLysAspSerGlyAspAsnVal 115120125 AsnAlaTrpGluSerGlySerAsnThrGluAspValLeuGlyLeuGly 130135140 ThrIleGlyArgValGluSerArgGluIleSerValArgTyrAspSer 145150155160 ProValPheAlaGlyPheSerGlySerValGlnTyrValProArgAsp 165170175 AsnAlaAsnAspValAspLysTyrLysHisThrLysSerSerArgGlu 180185190 SerTyrHisAlaGlyLeuLysTyrGluAsnAlaGlyPhePheGlyGln 195200205 TyrAlaGlySerPheAlaLysTyrAlaAspLeuAsnThrAspAlaGlu 210215220 ArgValAlaValAsnThrAlaAsnAlaHisProValLysAspTyrGln 225230235240 ValHisArgValValAlaGlyTyrAspAlaAsnAspLeuTyrValSer 245250255 ValAlaGlyGlnTyrGluAlaAlaLysAsnAsnGluValGlySerThr 260265270 LysGlyLysLysHisGluGlnThrGlnValAlaAlaThrAlaAlaTyr 275280285 ArgPheGlyAsnValThrProArgValSerTyrAlaHisGlyPheLys 290295300 AlaLysValAsnGlyValLysAspAlaAsnTyrGlnTyrAspGlnVal 305310315320 IleValGlyAlaAspTyrAspPheSerLysArgThrSerAlaLeuVal 325330335 SerAlaGlyTrpLeuLysGlnGlyLysGlyAlaGlyLysValGluGln 340345350 ThrAlaSerMetValGlyLeuArgHisLysPhe 355360 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 187 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 101..187 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC60 TAGAAATAATTTTGTTTAACTTAAAGAAGGAGATATACATATGGCTAGCATGACT115 MetAlaSerMetThr 15 GGTGGACAGCAAATGGGTCGGGATTCAAGCTTGGTACCGAGCTCGGAT163 GlyGlyGlnGlnMetGlyArgAspSerSerLeuValProSerSerAsp 101520 CTGCAGGTTACCTTGTACGGTACA187 LeuGlnValThrLeuTyrGlyThr 25 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: MetAlaSerMetThrGlyGlyGlnGlnMetGlyArgAspSerSerLeu 151015 ValProSerSerAspLeuGlnValThrLeuTyrGlyThr 2025 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTTGGTCTGCGTCACAAATTCTAACTCGAGCAGATCCGGCTGCTAACAAAG51 ValGlyLeuArgHisLysPhe 15 CCC54 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ValGlyLeuArgHisLysPhe 15 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGGTAGATCTGCAGGTTACCTTGTACGGTACAATTAAAGCAGGCGT47 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGGGGGTGACCCTCGAGTTAGAATTTGTGACGCAGACCAAC42 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCAAGCTTGGTACCGAGCTC20 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTTGTTAGCAGCCGGATCTG20 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTCAAGACCCGTTTAGAGGCC21 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGCGGCTTGGAATTCCCGGCTGGCTTAAATTTC33 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CAAACGAATGAATTCAAATAAAAAAGCCTG30 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCTGTTGCAGCACATATGGACGTTACCTTGTACGGTACAATTAAAGC47 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CGACAGGCTTTTTCTCGAGACCAATCTTTTCAG33 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCTGTTGCAGCGGATCCAGACGTTACCTTGTACGGTACAATTAAAGC47 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGACAGGCTTTTTCTCGAGACCAATCTTTTCAG33 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTTGCAGCACATATGGACGTTACCCTGTACGGCACC36 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGGGGATGGATCCAGATTAGAATTTGTGGCGCAGACCGACACC44 __________________________________________________________________________